Cover Page. The handle holds various files of this Leiden University dissertation.

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1 Cover Page The handle holds various files of this Leiden University dissertation. Author: Luo, Jinghui Title: Natural and non-natural factors influencing Alzheimer s Aβ Issue Date:

2 Part II The mechanism of amyloid fibrillation 49

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4 Chapter 4 Alzheimer peptides aggregate into transient ±100 nm cytotoxic globules that nucleate fibrils Jinghui Luo 1, Sebastian K.T.S. Wärmländer 2, Astrid Gräslund 2 *, Jan Pieter Abrahams 1 * 1, Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2333 CC, Leiden, The Netherlands 2, Department of Biophysics and Biochemistry, Stockholm University, Svante Arrhenius väg 16C, SE Stockholm, Sweden *Corresponding author: astrid@dbb.su.se, abrahams@chem.leidenuniv.nl Submitted to Biochemistry (in revision) 51

5 fibrils Abstract Protein/peptide oligomerisation, cross-β strand fibrillation and amyloid deposition play critical roles in many diseases(1 4) but despite extensive biophysical characterization(5 7), the structural and dynamic details of oligomerization and fibrillation of amyloidic peptides/proteins remain to be fully clarified(8). Here, we simultaneously monitored the atomic, molecular and mesoscopic states of aggregating Alzheimer s amyloid β (Aβ) peptides over time, and determined the cytotoxicity of the intermediate states. We show that in the early stage of fibrillation (the lag phase(3, 9)) the Aβ peptides coalesced into apparently unstructured globules ( nm diameter), which slowly grew in size. Then a sharp transition occurred, characterized by the first appearance of single fibrillar structures of about 100 nm or longer that grew from the globules, and the emergence of the cross-β strand conformation characteristic of the fibrils that constitute mature amyloid. The single fibrils rapidly increased in number and size. Eventually the fibrils coalesced into mature amyloid. Samples from the early lag phase were especially toxic to cells, and this toxicity sharply decreased with the formation of fibrils and their maturation into amyloid. Our results suggest that the formation of fibrils may protect cells by reducing the toxic globules that appear in the early lag phase of fibrillation. 4.2 Introduction In many diseases, amyloidogenic proteins including the Alzheimer s Disease related Aβ40 and Aβ42 peptides, but also the islet amyloid polypeptide, α-synuclein, prion protein and insulin assemble into disease-associated cross-β fibrils(10 12). Before and/or during the formation of fibrils, the peptides or proteins have to convert into a β-sheet structure. Prior to combining into amyloid material, monomeric peptides have been observed to form aggregates of diverse morphologies, indicating complex aggregation pathways. For instance, recent evidence reveals that fibrils of the human Aβ peptide can exist in several distinct and persistent structural variants: preformed seeds determine the structural variant of the fibril(13). Similar observations have been reported for prion proteins(10). Amyloid fibrillation includes a lag phase, a transition phase and a saturation phase(3, 9). These stages also characterize crystallization, strongly suggesting that the formation of stable nuclei from which the fibrils grow, is a rate-limiting step. During fibrillation, unfolded or partially unfolded proteins associate into small, soluble prefibrillar oligomers (PFOs) and fibrillar oligomers (FOs), which eventually develop into mature fibrils(1, 3,

6 fibrils 53 14). PFOs and FOs are morphologically and immunologically distinct and specific antibodies can discriminate between PFOs, FOs, and annular protofibrils(15). Importantly, PFOs have been shown to be more toxic than FOs(16, 17). FOs were reported to nucleate spontaneously from the monomeric state and are recognized by anti-fibril antibodies. FOs may represent fibril nuclei or small pieces of fibril that grow into fibrils by capturing soluble monomers at their ends(15). It is not clear whether the growth of FOs requires the presence of PFOs, or what role PFOs play in fibril formation. It has been proposed that PFOs are cylindrical β-barrels, in contrast to the linear β sheets of the FOs. This theory is based on an analogy with the behavior of the alpha-b crystallin chaperone protein, which can exist as aggregated amyloid or as a hexameric β-barrel. In this latter structure, the inter-strand hydrogen bonds between the β strands pair different residues compared to the residues paired in the amyloid form. It was hypothesized that such barrel structures are similar to the more toxic structures of the Aβ peptide that precede amyloid formation(16, 17). Although this hypothesis remains to be proven, it fits reasonably well with our observations, and we will base some of our interpretations on it. Besides the structures mentioned above, many more intermediate complexes have been reported, and the nomenclature and understanding of all these intermediate complexes is still developing(18). There are many reports interpreting the secondary structure, toxicity and morphologies of Aβ aggregates in specific phases of fibrillation. For instance, Dahlgren et al reported that Aβ42 oligomers are ten times more toxic to neuronal cells than fibrils, and even 40-fold more than toxic than the unaggregated peptide(19). Another early study showed that Aβ40 aggregates first form large-size aggregates, that subsequently convert into small-size aggregates, although little information was provided about these aggregates(20). We also investigated the structural transition and fibrillation of Aβ in the absence and presence of different ligands (such Gramicidin S(21), polyamines(6) and lysozyme(7)). A limiting factor of these studies is however the difficulty of properly correlating the different biochemical and biophysical properties of Aβ aggregates to the different kinetic stages of amyloid fibrillation, due to the heterogeneity and instability of the Aβ structures. The aim of the present study is therefore to simultaneously characterize (i.e. using one single sample that is simultaneously analyzed with different techniques) both structure and function of the Aβ peptide, as its aggregation evolved through the lag-, transitionand saturation phases. For this purpose we combined existing methods into a more comprehensive approach for investigating Aβ fibrillation. This approach has allowed us for the first time to accurately correlate the molecular structure, toxicity and morphology of Aβ aggregates in the different phases of fibrillation. This approach allowed us to

7 fibrils 54 visualise the appearance of oligomer aggregates preceding amyloid fibrillation and the subsequent nucleation of Aβ fibrils on these aggregates. 4.3 Methods & Materials Aβ(1 40) Peptide Preparation The amyloid-β peptide Aβ(1 40) and Aβ(1 42) were purchased either unlabeled or 15N-labeled from AlexoTech AB (Umeå, Sweden) and prepared according to previously described protocols(6) Setup 1: Circular Dichroism (CD) spectroscopy and Atomic Force Microscopy (AFM) based on Thioflavin T (ThT) Fluorescence Assays A 10 mm of ThT stock solution was prepared in dh2o. To aliquots of this solution ThT was added in the required amounts, and finally freshly prepared Aβ(1 40) peptide was added, yielding final samples containing (in 2ml): 5 µm ThT, 10 µm Aβ(1 40), 10 mm phosphate buffer(ph7.2). The sample was pipetted into a 10mmσ10mm path-length quartz cuvette with a plastic cap. The sample was incubated at 37 C, with mechanical stirring in a Jobin Yvon Horiba Fluorolog 3 (Longjumeau, France). The excitation and emission wavelengths of 446 and 490 nm, respectively, were recorded during measurements. The excitation and emission slits were set at 2 nm and 1nm, respectively. The fluorescence data were obtained in a time interval of 5 seconds and fitted to calculate the lag time and transition time using a Boltzmann function. During fluorescence measurement, the cuvette was temporarily and quickly transferred to a CD spectrometer (a Chirascan CD unit from Applied Photophysics, Surrey, U.K.) Far-UV CD spectra were recorded at 37 C, between 190 and 260 nm using a bandwidth of 1.0 nm. Immediately after the CD measurements, the cuvette was immediately returned to the Fluorolog for continuing thetht fluorescence assay, ensuring the orientation of the cuvette was always the same. After each CD measurement, 10 µl of sample was quickly transferred from the cuvette and deposited on freshly cleaved mica for 3mins for AFM measurement. After 3 min, excess liquid on the mica was shaken off, and the mica plate with deposited sample was rinsed once with 10 mm phosphate buffer (ph7.2) and dried in a stream of dry air at room temperature. Specimens were mounted on a Multi-Mode atomic force microscope

8 fibrils 55 (Digital Instruments Nanoscope III), and images were collected in tapping mode at 70 khz. The imaging was carried out in air, using silicon cantilevers with an asymmetric tip and a force constant of 3 N/m NMR assay and cell toxicity based on ThT and CD experiements A Bruker Avance 700 MHz spectrometer with cryogenic probe was used to record 1H- 15N HSQC spectra at +5 C of 500 µl 90 µm 15N-labeled Aβ(1 40) peptide in 20 mm sodium phosphate (ph 7.2 ), with 45 µm ThT at (90/10 H2O/D2O). Afterwards, the sample was taken out and incubated in an Eppendorf tube for 0.5 h at 20 C and put back to NMR tube for recording the next NMR spectrum. The same procedure and conditions were repeated after 1.7, 3.2 and 5.7 hours, respectively. The data were analyzed using the Sparky software ( After each incubation, 10 µl of the sample was quickly transferred out from the Eppendorf tube into 90 µl of 20 mm sodium phosphate buffer (ph 7.2) for CD and ThT fluorescence assays in a 0.5mmσ10mm cuvette (see above). Separately, we prepared samples an identical fashion for cell toxicity assays. Neuroblastoma SH-SY5Y cells were used with a maximum passage number of 15. Cells were cultured to a confluency of 85% in a 75 cm2 flask (Greiner Bio-one, cat ), in Dulbecco s modified eagle medium (a 1:1 mixture of DMEM and Ham s F12 medium) and 10% supplemental fetal bovine serum, containing 1% (vol/vol) penicillin/streptomycin at 37 C, 5% CO2. Cells were detached by 5 mm EDTA/PBS for 5 min at 37 C. Then cells were resuspended at a concentration of cells/ml in DMEM/F12, containing 1% (vol/vol) penicillin/streptomycin. The resuspended cells were plated at a volume of 50 µl and a cell density of cells/well in a 96-well plate. The plated cells were incubated for 48 h at 37 C at 5% CO2. Aβ40 oligomer-enriched fractions were prepared at a concentration of 90 µm after 0, 1.7, 3.2, 5.7 and 21 hours incubation at 20 C (identical as used for the NMR, ThT and CD measurements, see above). The Aβ aggregates were diluted to a final concentration of 25 µm when added to the cell cultures. The phosphate buffer as a control was added at a volume of 50 µl in medium to each well and incubated for 48 h. After 48 h, the plate was equilibrated at room temperature for approximately 30 minutes. CellTiter-Glo R Luminescent Cell Viability Assay (Promega, cat. G7571) compound was added to each well and the plates were agitated on an orbital shaker for 2 minutes to induce cell lysis. Luminescent intensity was measured on a 384-well plate reader (Infinite M1000 PRO microplate reader) at 1000 ms integration time. Data were measured in three independent experiments and statistical analysis was used for calculating the reported average values and standard deviations.

9 fibrils Results & Discussions Figure 1: Synchronous measurements of Aβ40 fibrillation by (a) ThT fluorescence and (b) CD spectroscopy. The transition of secondary structure of Aβ40 was measured in parallel on the same sample and within the same cuvette using two independent assays. Note the slow conversion from random to beta strand during the first 50 minutes, around an isodichroic point at 208 nm in the CD spectrum, followed by a rapid transition between 50 and 80 minutes. (c) The CD intensities at 195 nm and 218 nm plotted vs time. We followed the kinetics of the formation of the Aβ40 peptide cross-β structures that characterize amyloid fibrils using a ThT assay. A lag time of 50 minutes during which no cross-β structures were formed, was followed by an exponential rise in fluorescence for about 20 minutes (indicating amyloid fibrils were being formed rapidly) and a linear phase lasting about 20 minutes. Thus, the transition phase lasted around 40 min, and after about 200 minutes a steady-state was reached (fig.1). A similar, albeit faster, behavior was observed for the related the Aβ42 peptide (see supplemental information fig.s2). These kinetic ThT results follow the typical pattern for Aβ aggregations, and were confirmed by simultaneous CD spectroscopy of the same sample (measured in the same cuvette). The CD spectra are generally characterized by a two-state transition from random coil to beta-sheet structure, with an isodichroic point around 208 nm. This transition is slow at first, but becomes fast after about 50 minutes (fig. 1b). This time point coincides with the end of the lag phase we observed in the ThT assay. The initial small increase in β-strand conformation could have been caused by the formation of PFOs, which according to a recently proposed hypothesis are are β-barrel type structures(15 17, 22). ThT intercalates between the β-strands in the fibril(23), but there is no indication that it binds β-barrels, which would explain why we did not observe an in ThT fluorescence in the early stages. The rapid structural shift from random coil to β strand observed in the CD spectra between minutes (fig.1b), i.e. during the ThT transition phase, is striking and indicates that fibrils can grow quickly once the FOs have nucleated. Interestingly, the CD spectra at time points 64 and 71 minutes are off the isodichroic point. Such offset is typically due to light scattering effects caused

10 fibrils 57 by the presence of large aggregates, and the AFM results (below) indeed confirmed the presence of transient large globules during the transition phase. Figure 2: (a-c): The relative intensity of NMR cross-peaks after incubating at 20 C for 0.5 h (a), 1.7h (b) and 3.2h (c), respectively. Parallel measurement of changes in secondary structure of the NMR sample using a fluorescence ThT assay and CD spectroscopy. (e): The secondary structures of Aβ40 after 0h (red), 0.5h (blue), 1.7h (green) and 3.2h (cyan) incubation. (f): Cell toxicity measurements of identical samples indicate that the most toxic species is formed early in the incubation and that over time the samples become less toxic. NMR spectroscopy measurements confirmed the conformational changes of Aβ40 during the early lag phase (fig. 2, fig. S4). For the NMR studies, Aβ aggregation was slowed down by incubating in an Eppendorf tube at room temperature instead of +37 C, between the NMR measurements which were carried out at 5 oc. The reduction in incubation temperature prolonged the lag phase to about 5 hrs (CD and ThT data not shown), while reducing the temperature to 5 oc (required for the NMR measurement) completely stopped the aggregation process. An overall reduction in Aβ amide HSQC

11 fibrils 58 cross-peak intensity was observed as the incubation progressed (fig.2a-c). This signal loss can be attributed to formation of larger peptide aggregates, which are in exchange with the monomer on a slow or intermediate timescale(24). The observed NMR signal originates only from the Aβ monomer and possibly low-molecular-weight aggregates. After 30 minutes about 25% of the original cross-peak intensity remained, whereas after 100 minutes about 40% of the initial signal intensity was observed. After 200 minutes only 20% of the signal intensity remained. While the signal intensity is expected to decrease over time due to aggregation, the initial intensity drop after 30 minutes followed by a signal recovery after 100 minutes was reproducible and unexpected (fig. S5). These intensity changes are most likely related to changes in the chemical exchange, as exchange on intermediate time-scales can induce extensive loss of NMR signal, while slow exchange usually has more moderate effects. Thus, the loss and reappearance of NMR signal intensity are arguably linked to chemical exchange with a PFO species that first grows and then disappears by converting to a FO form it is previously known that PFOs and FOs have different conformational features(14). As the NMR intensity signal effect roughly correspond in time with the appearance and disappearance of large globular aggregates (see AFM results below), these globules are strong candidates for the intermediate chemical exchange species. No significant chemical shift changes were observed, showing that the Aβ monomer population largely remains in its initial random-coil like structure. We observed the changing morphology of the Aβ40 aggregates at the mesoscopic scale by AFM. During the lag phase, small, spherical aggregates were observed, which grew or coalesced over time (fig.3) in a process that may compare well to classical phase separation, but at a much smaller scale. This suggests that the initial conformational change of the Aβ peptide results in the formation of a relatively more hydrophilic complex. Three processes that do not exclude each other can explain the increase in size of the spherical aggregates. (i) Monomeric peptides from solution are converted into PFOs and then condense on the globular aggregates. (ii) The PFOs trapped in aggregates may be in dynamic equilibrium with the solution, allowing PFOs to exchange between globular aggregates. The larger an aggregate, the smaller the chances of PFOs escaping (because of the decreased surface-to-volume ratio), causing large aggregates to grow at the expense of smaller ones. (iii) Globular aggregates may fuse to form bigger aggregates. After about 60 minutes fibrils started growing from the larger globular aggregates, and they continued to grow. Close inspection of the AFM images revealed that the fibrils stuck to the surface of the nano-spheres, as we frequently observed the fibers lying over of these otherwise featureless aggregates. After 340 minutes, well into the plateau observed using the ThT assay, the spheres were no longer visible and only fibrillar aggregates could be seen. The formation and disappearance of these large spheres explain

12 Alzheimer peptides aggregate into transient ±100 nm cytotoxic globules that nucleate fibrils 59 Figure 3: Aβ40 fibrillation observed by atomic force microscope, of the samples that were simultaneously studied by ThT fluorescence (fig 1.a) and CD spectroscopy (fig 1.b). (a-f): AFM images of Aβ40 from the different phases of fibrillation as monitored by a by ThT fluorescence assay (fig 1.a), Aβ40 samples from the cuvette of fluorescence assay were taken after 20mins (a), 40mins (b), 57mins (c), 71mins (d), 91mins (e), 340mins (f) for AFM measurements. The size of black scale bars is 1 µm. (g-l) : The size distribution as defined by the ratio between the perimeter length distribution versus the volume of aggregates corresponding to the AFM measurements at the given time points (a-f).

13 fibrils 60 Figure 4: Summary of the changes to the secondary structure, cell toxicity and morphology of Aβ oligomers as they pass through different phases of amyloid fibrillation. There are three different phases in amyloid fibrillation: the lag, transition and saturation phase. In the lag phase, the fibrillation pathway starts with a disordered monomer and can diverge into two directions to form aggregate variants which we also observed by AFM images. The aggregates show a steep decrease in toxicity in the transition phase. The secondary structure of amyloid β peptide converts from random to β-sheet when passing from the lag phase to the saturation phase and has an intermediate structure during the transition phase. Tjernberg et al. s observation from 1999 that Aβ first form large aggregates that later convert into small ones(20). The spheres arguably also explain why CD spectra during the transition phase deviate from the isodichroic point (the large spheres induce light-scattering), and may also be related to the loss and reappearance of NMR signal intensity (via intermediate chemical exchange). The free ends of the fibrils were hardly ever associated with the nano-spheres, indicating that growth of the fibers occurred in solution by capturing soluble Aβ peptides. The eventual disappearance of the globular aggregates must therefore result from the dissolution of peptides from these globules followed by accrual at the free fibre-ends, and not by their direct incorporation from of the aggregated state into the growing fibres In conclusion, our data therefore strongly suggest that growth of the fibrils occurs at both ends and does not involve a direct transfer of mass from peptide in the globular aggregate to the fibrillar state. Our results indicate that the Aβ40 peptides can be present in at least four different physico-chemical states: as soluble monomers, as globular prefibrillar oligomeric aggregates, in a fibril and as mature amyloid material (fig. 4). Initially peptides coalesce

14 fibrils 61 into apparently unstructured aggregates. We assume these aggregates to be enriched in PFOs. Extensive conversion of the random coil structure to β sheet conformation is not yet occurring at this stage, as our CD data indicate, although some β sheet may be forming. Our results indicate that the formation of fibrils requires a (rare) nucleation event that takes place in or on the globular aggregates. As we observe the fibrils to form more readily on large, rather than small globular aggregates, we assume that fibril nucleation site is a stochastic process: the larger the aggregate, the higher the chances of nucleation. Then the fibrous phase (characterized by the conversion of peptides into a β strand conformation and the formation of cross-β sheets) emerges, marking the start of the transition state. The fibrils bundle into mature amyloid in the saturation state. One of the motivations of our studies was to relate these physical changes of the Aβ peptide to toxicity. As it is likely that the early aggregates are more toxic than the late, mature fibrils, we again incubated the Aβ peptide at 20 C just as in the NMR experiment. We took samples at the same time points as in the NMR experiments and added them to growing neuroblastoma SH-SY5Y cells (fig.2p). In parallel, we verified the aggregation state of the same batch of sample with a ThT assay (Fig.S3). We observed no effect on cell viability if the monomeric Aβ was added directly to the cells, without being allowed to start aggregating. However, 30 min into the lag phase, which is characterized by the formation of PFOs, cell viability was severely affected, indicating that especially in the early stages of Aβ aggregation, the most toxic forms are produced (and that the medium within which the cells were cultured, slows down Aβ aggregation). When aggregation was allowed to progress before adding the sample to the growing cells, cell viability increased again, indicating that the later-stage aggregates, in which fibrillar structures could be recognized in AFM, were less toxic or protected the cells. If the PFO barrels would indeed have a relatively hydrophobic surface as suggested by our data, they might be able to insert into cell membranes, similar to the action of pore-forming toxins(25). Possibly, it is the hydrophobic aggregate itself that also contributes to the toxicity of the peptide. In order to prevent neuronal damage that is associated with Aβ fibrillation, it may be advantageous to look for inhibitors of PFO formation and/or aggregation of PFOs, as this would reduce the most toxic structural species. Alternatively, and perhaps somewhat paradoxically, it may also be advantageous to investigate molecules that can speed up the conversion of PFOs into FOs.

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17 Alzheimer peptides aggregate into transient ±100 nm cytotoxic globules that nucleate fibrils Supplementary information Figure S1: Independent re-measurement of oligomerisation/aggregation behavior of Aβ40 by ThT and CD fluorescence spectroscopy (repeat of fig. 1 see the legend of fig. 1 for details) shows that though the overall behavior is reproducible, there are subtle differences between samples, necessitating careful parallel measurements. The transition of secondary structures of 10 µm Aβ40 sample in ThT fluorescence assay (a) was simultaneously measured in same cuvette by CD spectroscopy (b). The CD intensities in the wavelength of 195 nm and 218 nm plotted vs time in the panel of c. Figure S2: Parallel oligomerisation measurements of the 2 aa longer, less stable Azheimer peptide Aβ42. Fibrillation was measured by atomic force microscope, ThT fluorescence and CD spectroscopy. a-d: AFM images of Aβ42 from the different phases of amyloid fibrillation as identified by ThT fluorescence and CD spectroscopy (e-g), Aβ samples were taken from the cuvette for CD and fluorescence assays and analysed by AFM. The samples were extracted after 5 min (a), 30 min (b), 60 min (c) and 150 min (d). The size of black scale bars is 1 µm. The repeat experiment (h-j) shows that also for Aβ42 though the overall behavior is reproducible, there are subtle differences between samples.

18 fibrils 65 Figure S3: ThT fluorescence assay of Aβ40 from the same batch sample that was used for the cell viability assay. Figure S4: Left: 1H-15N HSQC spectra of 90 µm 15N labeled Aβ peptide before (red peaks) and after incubation at room temperature for 0.5h (blue peaks), 1.7h(green peaks), 3.2h (cyan peaks) and 5.7h (purple peaks), show that there are no observable changes in chemical shift.

19 fibrils 66 Figure S5: The repeat NMR experiments show that the initial strong drop in NMR intensity, followed by a recuperation of the signal, is reproducible. The relative intensity of NMR cross-peaks after 0.5 h(a) and 1.7h(b) incubation at 20 oc is shown.